Project Summary Infertility is a widespread condition, which has affected more than 10% of all couples worldwide. For these couples, in vitro fertilization (IVF) represents the best chance to conceive. However, for IVF, the overall rate of a successful birth is less than 25%. Moreover, while most IVF children appear healthy, reports showed suboptimal exposures during pre-implantation in vitro culture can affect postnatal growth, glucose metabolism, fat deposition, and vascular function. It is generally believed that, compared with sperm-oocyte fusion in vitro (which needs less than 10 hours in human), several days of pre-implantation embryo in vitro culture is more responsible for these epigenetic disorders. This is not a surprise due to the drastic differences between in vitro and in vivo environment for the pre-implantation embryo. While over the past few decades, embryo culture media, incubation/observation systems, and oxygen level controls have been drastically improved, fine-control of the microenvironment of embryos has been overlooked. For example, culture of pre-implantation embryos still utilizes traditional 2D tissue culture polystyrene surfaces. Thus, in this project, we focus on improving the pre-implantation embryo culture by providing a biomimetic microenvironment. Our previous research has demonstrated the importance of biophysical cues in the fate decision of human embryonic stem cells. Based on preliminary studies, we hypothesize that optimal matrix stiffness, fluid flow, and embryo rolling will improve the quality of embryo culture in vitro. To accomplish our objectives, we will first interrogate the independent role of matrix stiffness, fluid flow and embryo rolling in embryo development (Aim 1). Further, to integrate these cues, we will develop a novel Actuatable Fibrillar Substrates (AFS) to mimic ciliated oviductal epithelial cells, as potential replacements for current IVF dishes. The evaluate the effects of AFS on embryo development, we will examine cell lineage markers, expression pattern of imprinted genes with their allele-specific DNA methylation, as well as differential effects associated with gender and cell types of embryo. Further, implantation frequency and fetus/placenta development in vivo will be tested using pseudopregnant mice (Aim 2). The long-term objective of this project is to advance the IVF procedures to improve the overall success rate and reduce postnatal diseases related to IVF procedures. Notably, the AFS proposed here are biocompatible, multi-functional, easy to use, and fully automated. It is compatible with embryos grown in culture medium droplets covered by an oil layer, which are routinely used in clinics. Real-time monitoring embryo growth using time-lapse incubators is also allowed with our platform. Thus, clinicians can use AFS the same way as standard IVF dishes with minimal hurdles of adaptation.